[unreadable] Ubiquitin-mediated protein degradation down-regulates hematopoietic regulatory proteins (e.g. AML-1, p130, p27, and JAK2). Importantly, dysregulation of AML-1 and JAK2 contributes to leukemogenesis. Therefore, determination of how ubiquitination regulates hematopoiesis at a molecular level is crucial for devising new methods to prevent or treat leukemia. The substrate specificity of ubiquitination is largely determined by ubiquitin ligases, but the specific ligases that target these regulatory proteins are unknown. The Cul-4A cullin is a ubiquitin ligase core subunit. Over-expression of CUL-4A in hematopoietic cell lines interferes with cell cycle exit and granulocyte and erythrocyte terminal differentiation. Nearly half of CUL-4A haploinsufficient mice (CUL-4A) are nonviable and die in utero. These embryos appear pale, suggesting that CUL-4A is required for proficient embryonic hematopoiesis. The remaining CUL-4A mice are viable but display abnormalities in adult hematopoiesis. The central hypothesis of this proposal is that CUL-4A is required for normal embryonic hematopoiesis. CUL-4A embryonic stem (ES) cells will be differentiated in vitro to assay embryoid body (EB) hemoglobinization. EB-derived progenitors will be assayed for their ability to form hemangioblast, primitive erythroid, and definitive hematopoietic colonies. Differentiation to mesoderm will also be examined (Aim1). To corroborate in vitro findings, primitive and definitive progenitors in the yolk sac and fetal liver, respectively, will also be assayed in CUL-4A embryos (Aim2). These aims will identify CUL-4A-dependent hematopoietic differentiation pathways, which will become the focus of future studies to determine the molecular mechanism for how the Cul-4A ubiquitin ligase regulates embryonic hematopoiesis. Dr. Chun will train with Dr. Yoder to learn the principles and methodologies for examining the in vitro differentiation of murine ES cells and murine embryonic hematopoiesis. With these skills, Dr. Chun will establish a model system that will facilitate future studies that include identifying the hematopoietic regulators that are degraded by a Cul-4A ubiquitin ligase [unreadable] [unreadable]